Apparatus for introducing biological material, method of introducing biological material and magnetic support for introducing biological material

ABSTRACT

The invention relates to an apparatus for introducing a biological material, a method of introducing a biological material, and a magnetic support for introducing a biological material with the object of providing an apparatus for introducing a biological material, a method of introducing a biological material, and a magnetic support for introducing a biological material whereby a biological material can be efficiently introduced into a host. 
     The invention comprises: one or more packing units in which a mixture solution containing a large number of magnetic supports carrying a biological material to be introduced into a host such as cells upon using, together with a large number of the hosts in a liquid is pooled; and an introduction treatment unit in which a magnetic force affecting the inside of the packing unit is controlled so as to move the magnetic supports relatively with respect to the host so that the biological material can be introduced into the host.

This application is a Continuation of copending application Ser. No.10/531,520 filed on Nov. 16, 2005, which is the National Stage Entry ofPCT/JP2003/013254 filed on Oct. 16, 2003, which claims priority toApplication No. 2002-301916 filed in Japan on Oct. 16, 2002. The entirecontents of all of the above applications is hereby incorporated byreference.

TECHNICAL FIELD

The present invention relates to an apparatus for introducing abiological material, a method of introducing a biological material, anda magnetic support for introducing a biological material. Specifically,the invention relates to an apparatus for introducing a biologicalmaterial, a method of introducing a biological material and a magneticsupport for introducing a biological material by which a geneticmaterial such as a foreign DNA can be introduced into a cell of anorganism or a bacterium using a magnetic support, so as to be utilizedin the various fields such as the manufacturing industry medical healthcare services, the pharmaceutical industry, apiculture, fishery,livestock industry, and biochemistry which perform DNA cloning, genetherapy, breeding of plants or animals, production of useful proteins,and biochemical analysis.

BACKGROUND ART

Conventionally, in the field of genetic engineering, in order to performDNA cloning, gene therapy, or breeding, a vector is necessary as acarrier which brings a foreign target gene into a cell of an organism,in addition to enzymes which cut and paste genes. If a bacterium such asE. coli as a host is used as such a vector, a plasmid or a λ phage hasbeen used and mixed with E. coli and the like, then electroshocked tointroduce this. Moreover, in order to separate the E. colis introducedin this manner, the process has been such that, after a series oftreatments to combine an antibiotic resistant gene resistant toantibiotics into the plasmid or the like, only cells which aretransformed through the antibiotic treatment are extracted.

Moreover, there have been three methods for bringing a foreign targetgene into an animal cell, namely; (1) to introduce a foreign gene as is(2) to introduce it by microinjection, and (3) to make a ret early it.

Introducing a foreign target gene as is, into an animal cell isperformed by mixing calcium phosphate with the foreign genes, and thenmixing the precipitated genes with the animal cells. The reason for thisis that by so doing even a small number of cells can take-in theprecipitated genes.

In the microinjection, a foreign gene is directly inserted into a cellnucleus using a micropipette (extra fine capillary tube) which is madefrom very fine glass having a diameter of 0.1 μm, while watching througha microscope.

Moreover, in older to use a retrovirus as a vector, a foreign targetgene is sandwiched between the opposite terminals by a retrovirus LTR(transcriptional promoter and poly A-binding site) to create the vector,which is then introduced into an animal cell.

Furthermore, there is another method wherein the biological material isintroduced using magnetic particles by shooting the magnetic particlesfixed with the biological materials into cells, organs, or tissues athigh speed (initial velocity is 50 to 400 m/second) (for example, referto Japanese Unexamined Patent Publication No. 6-133784, paras. 14, 19,and 20). This method is to facilitate the concentration or separation ofcells by using magnetic three.

Incidentally, in the conventional method of introducing a target genedescribed above, a parasitic genetic factor (extrachromosomal gene of abacterium which can proliferate independently from chromosomes and thelike) such as a plasmid or a virus such as a λ phage has been used as avector. There has been a problem in that, the parasitic genetic factorand the virus are intrinsically pathogenic in many cases, and even ifthe pathogenicity is put out of action in use, they may recover thepathogenicity by recombining with another pathogen infected to the host.Moreover, there has been another problem in that, even if electroshockis applied during the introduction, the introduction efficiency is nothigh enough. Furthermore there has been another problem in that, due tothe presence of the antibiotic resistant gene used when the introducedcells are extracted, the diffusion of the antibiotic resistant gene maynegatively affect the environment of the host.

If the host is an animal cell, the first method can be readily performedsince the operating procedure is simple. However, this method has aproblem of extremely poor efficiency. The second method allowsintroducing the target gene into the cell nucleus reliably. However,there has been a problem in that to master this technique requires areasonable technical training and takes time.

In the third method, a package cell strain having the autonomousreplication-competence deleted and genes required for proliferationcombined into a chromosome is used for ensuring the safety of theretrovirus. However, there has been a problem in that, similarly to theabovementioned plasmid, the retrovirus may recover the pathogenicity byrecombining with another pathogen infected to the host.

In the method of shooting the magnetic particles fixed with thebiological materials into cells at high speed using a firing apparatus,there have been problems in that the scale of the apparatus becomeslarger, and that the magnetic particles which are once fired and goastray from the cells can not be reused, and hence the efficacy is lowafter all. Them has been another problem in that since they are shot athigh speed, cells having low strength are broken down when the particlescollide with the cells, making it impossible to introduce the biologicalmaterials thereinto.

As described above, any method has a problem of poor efficacy, since theway to introduce a biological material into a host is to let the hostand the biological material suspended or contained in the solutionencounter naturally, or to introduce the biological material into thehost one by one manually, or only once by a firing apparatus.

Therefore, the present invention has been developed to solve theabovementioned problems, with a first object of providing an apparatusfor introducing a biological material, a method of introducing abiological material, and a magnetic support for introducing a biologicalmaterial, whereby a foreign biological material can be introduced into ahost efficiently, not by relying on a natural encounter of a host and abiological material, but by using a magnetic force and controlling itsmagnitude, direction, location, and the like, to promote the collisionby moving the magnetic support until it enters the host, or to promotethe encounter by densifying the biological material in the solution.

A second object is to provide an apparatus for introducing a biologicalmaterial, a method of introducing a biological material, and a magneticsupport for introducing a biological material, wherein varioustreatments are standardized and automatically performed without relyingon manpower nor skilled technicians, so that the introduction into ahost can be readily performed.

A third object is to provide an apparatus for introducing a biologicalmaterial, a method of introducing a biological material, and a magneticsupport for introducing a biological material, whereby the introductioncan be performed without using a recombinant of a virus or a parasiticgenetic factor such as a plasmid, and a biological material can beintroduced safely.

A fourth object is to provide an apparatus for introducing a biologicalmaterial, a method of introducing a biological material, and a magneticsupport for introducing a biological material, whereby a host having aforeign biological material introduced thereinto can be readily andreliably separated and extracted.

A fifth object is to provide an apparatus for introducing a biologicalmaterial, a method of introducing a biological material, and a magneticsupport for introducing a biological material, whereby the effect on theenvironment caused by combining an antibiotic resistant gene ma beavoided, enabling safe separation and extraction.

A sixth object is to provide an apparatus for introducing a biologicalmaterial, a method of introducing a biological material, and a magneticsupport for introducing a biological material, winch has variety andgenerality allowing introduction under various conditions according tothe properties of hosts and biological materials.

DISCLOSURE OF THE INVENTION

In order to solve the abovementioned technical problems, a first aspectof the present invention provides an apparatus for introducing abiological material which has: one or more packing units in which amixture solution containing a large number of magnetic supports carryinga biological material to be introduced into a host such as cells uponusing, together with a large number of the hosts in solution is pooled;and an introduction treatment unit which controls a magnetic forceaffecting the inside of the packing unit so as to move the magneticsupports relatively with respect to the host in solution to introducethe biological material into the host.

“Host” means an object into which the foreign biological material isintroduced, or is to be introduced, corresponding to for example,eukaryotic cells such as plant or animal cells (including human cell),prokaryotic cells such as bacteria, proteins, and the like.

“Biological material” is a material for giving desired properties to thehost, and includes for example, genes, enzymes, antigens, antibodies,proteins, pheromones, allomones, mitochondria, viruses, plasmid, and thelike.

The magnetic support carries the biological material by for example,physical adsorption, electrical interaction, chemical absorption,covalent binding, or the like.

“Magnetic support” is a support which is magnetic due to overall orpartial magnetization, or which is magnetizable. Its properties such asshape, size, and susceptibility, and the amount, and density redetermined by the method of introducing a biological material, and theproperties, the amount, and the density of the host, and the like. Thematerial of the magnetic support is for example a superparamagneticsingle-domain particle having a surface stabilizing substance. Theparticle is made of for example, an iron hydroxide, an iron oxidehydrate, an iron oxide, a mixed iron oxide, or iron.

The size of the magnetic support is determined by conditions such as thetype or the size of the host, or the type or the size of the biologicalmaterial. For example, if the host is a prokaryotic cell, it would beabout between 100 nm and 200 nm. If it is a eukaryotic cell such as forexample, yeast or an animal cell, it would be between 1 μm and 2 μm.

If the biological material is introduced by adhering the magneticsupport to the host, the shape of the magnetic support is globular forexample, and the particle surface coating substance is required to be amaterial which has a good adhesion onto the surface of the host.Furthermore, in the case where the host can not be adhered, the cellmembrane of the host is strong, or there is a cell wall, or in the casewhere the introduction into the host is performed without performingcell surface treatment, the magnetic support preferably has a shape withtapered ends.

“To control the magnetic force affecting the inside of the packing unit”means to control the direction or the size of a predetermined magneticforce affecting the inside of the packing unit, the relative position ofthe magnetic source with respect to the packing unit or the solution therelative speed or the acceleration of the magnetic source with respectto the packing unit or the solution, or the time to apply the magneticforce, so as to relatively move between the host and the magneticsupport, to promote collision, thereby increasing the number ofcollisions or the rate of collision (average number of collisions perunit time), or for example, so as to move the magnetic support carryingthe biological material with respect to a perforated host or a hostwhich has a soft boundary not requiring migration treatment, to make anarea where the biological material is densified mom than thesurroundings, thus increasing the chance of encounter with the host, andenabling to increase the introduction efficiency.

In order to promote collisions or encounters, the relative movementdistance of the magnetic support with respect to the host or thesolution is preferably longer. For example, reciprocating rectilinearmotion, rotation, vibration, or movement in a combination thereof, orperiodic movement such as movement along other closed curves, oraperiodic repetitive movement, are preferable.

For this purpose, the magnetic three is applied not only from onedirection, but also from at least two directions with the packing unittherebetween, and the magnitude, the direction, or the position thereofcan be preferably controlled independently. The reason for this is suchthat, if the magnetic force is applied only from one direction, themagnetic supports are moved only in one direction and applied with themagnetic fare for a very short time, and then they are collided with thewall of the packing unit along the direction, coming into a state wherethey are attached to or aggregated on the wall. Once the magneticsupports come into a state where they are attached to or aggregated onthe wall, the magnetic supports can not be released from the attachmentstate simply by removing the magnetic force. In such a state, it isdifficult to promote collisions or encounters with the hosts which arewidely scattered in solution or are aggregated in a part of thesolution. In order to separate the magnetic supports in the attachmentstate to increase the chance of collision or encounter with the host inthe solution, the magnetic force is applied in a direction to releasethe attached magnetic supports, for example in the opposite directionwith the packing unit therebetween, against the direction of themagnetic force which makes the magnetic supports become attached,thereby enabling the magnetic supports to pass through the approximatecenter of the packing unit and be moved in the opposite direction. Thechance of collision or encounter with the host is increased by repeatingthe above procedure. At this time, there where the magnetic force isapplied from another direction after removing the magnetic force whichmakes the magnetic supports become attached, or where a magnetic forceis added without removing the magnetic force.

Moreover, it is also possible to initially avoid or reduce theattachment of the magnetic supports onto the Wall of the packing unit soas to increase the chance of collision or encounter. For example, beforethe magnetic supports are attached onto the wall of the packing unit dueto the magnetic force applied from one direction, they are reciprocatedby applying a magnetic force from a different direction from the abovedirection. Furthermore, for example, magnetic forces having similarstrengths are horizontally applied from at least two directions at thesame time so that the combined magnetic forces applied to the magneticsupport counterbalance each other or are reduced as little as possible,in the horizontal plane. Then, in the state where the magnetic supportsare developed in the solution, the position where the magnetic force isapplied is vertically reciprocated or rotated at the same time, so thatthe magnetic supports are vertically reciprocated or rotated.

By controlling as above, the chance of collision or encounter betweenthe magnetic supports and the host can be increased more efficiently.

In this manner by moving the magnetic supports relatively with respectto the host in the solution by the magnetic force; by moving themagnetic supports relatively with respect to the host by releasing themagnetic supports from the attachment state by the magnetic force ifthey are attached onto the packing unit; by moving the magnetic supportsrelatively with respect to the host by initially avoiding a conditionwhere the magnetic supports are aggregated due to the attachment causedby the collision with the packing unit; or by moving the magneticsupports relatively with respect to the host in a state where themagnetic supports are actively developed in the solution, the chance ofcollision or encounter between the magnetic supports and the hostsuspended in the solution can be increased.

Here, “two directions” mean the directions orientation intoconsideration. If the orientation is different the direction is alsodifferent “Apply the magnetic force from at least two directions withthe packing unit therebetween” does not only mean a case where twodirections are facing linearly with the packing unit therebetween, butalso includes a case where they are facing at a certain angle with thepacking unit therebetween. Moreover, it does not only mean a case wherethe combined magnetic foxes applied to the respective magnetic supportscounteract each other, but also includes a case where the combinedmagnetic force is limited. Furthermore, it does not only mean a casewhere the magnetic forces in the respective directions are applied atthe same time, but also includes a case where they are applied atdifferent times. The magnitudes of the magnetic fortes in the respectivedirections are not limited to be the same. Since the magnetic force isapplied to the packing unit, the direction of the magnetic force isoriented to pass through the inside of the packing unit. The magneticforces in the respective directions are preferably related so as tocontrol the magnitude, the direction, and the position. The magneticforces are preferably controlled to be applied to the packing unit frommany directions, so that the magnetic supports are moved around ordistributed to develop in the solution.

The encounter of the magnetic support and the host means that themagnetic support and the host are in contact without perforating thehost. Accordingly, the magnetic support is adhered to the host, or themagnetic support is made to enter the host. On the other hand, thecollision of the magnetic support with the host means that the magneticsupport and the host are in contact with perforation of the host.Accordingly, the magnetic support is adhered to the host or entered intothe host with perforation. Therefore, in the introduction of thebiological material by the encounter, depending on the property of thehost, it is preferable to facilitate the introduction of the biologicalmaterial by electroporation or the like, that is, to have the functionof perforating the host. The packing unit is preferably formed of forexample a glass, or a plastic such as polyethylene, polypropylene, oracrylic resins, being transparent or semitransparent.

In order to promote the encounter, for example, the magnetic supportsare aggregated in a certain area in the solution in the direction of themagnetic force, to densify the area with more biological materials thanthe other areas. Such densification is effective in the case of aperforated host, or the case where the magnetic support enters a hostfor which perforation is not required for the magnetic support to enteraccording to its property.

According to the first aspect of the invention, by using the magneticsupports, the biological material can be mechanically or physicallyintroduced without using a vector such as a virus by using the magneticsupports, so that the biological material can be introduced safely.Between the magnetic supports caning the biological material and thehost in solution is relatively moved, not by relying on a naturalencounter or collision with the host, but by controlling the magneticforce, to increase the number of collision or the rate of collisionbetween the magnetic supports and the host, or to increase the chance ofencounter with the host, so that the introduction of the biologicalmaterial into the host can be promoted. Moreover, by using the magneticsupports having rigidity and mass, the impelling force is increased andmovement is facilitated in the solution, more than when using thebiological material only, so that the chance of collision or encounterwith the host is increased, facilitating the introduction into the host.

Moreover, since the introduction is performed by controlling themagnetic form, then by optimizing the manner of collision such as themagnitude, the velocity, and the acceleration due to the magnetic forceaccording to the properties of the host or the biological material, thehost can be kept from being broken down, and a highly densified area ofthe magnetic supports can be made, or the magnetic supports are freelymoved to the highly densified area of the host, so as to increase thechance of encounter. Moreover by combining in the above manners ofcollision, introduction having generality, variety, certainty,reliability, and efficiency can be performed.

A second aspect of the present invention is an apparatus for introducinga biological material, wherein the introduction treatment unit has: amagnetic source which can apply a magnetic force to the inside of thepacking unit; and a magnetic force control unit which controls themagnetic supports to move relatively with respect to the host, bychanging the relative position or the velocity between the packing unitor the mixture solution and the magnetic source, or the magnetic forceitself due to the magnetic source.

Since the movement is “relative”, the magnetic supports are relativelymoved with respect to the host such that: the host or the mixturesolution containing the host is made static or approximately static andthe magnetic support is moved; the magnetic support is made static orapproximately static and the mixture solution containing the host ismoved; or the mixture solution containing the host and the magneticsupport are both moved in different manners. If the host is a biologicalmaterial such as a cell with low strength, the magnetic support ispreferably moved. In order to avoid or reduce the collision of themagnetic supports onto the packing unit, the magnetic force is appliedfrom at least two directions with the packing unit therebetween. Since“the position or the velocity is changed”, uniform motion, acceleratedmotion, oscillation, rotation, and the like are included. “Velocity” isnot only the magnitude of the speed but includes its direction.

The magnetic source is permanent magnets or electromagnets movablyarranged around the packing unit, or fixed electromagnets arrangedaround the packing unit. The magnetic force control unit is a magneticthree transfer unit which relatively moves the magnetic source withrespect to the packing unit, or a magnetic force modification unit whichmodifies the magnitude of the magnetic force of the electromagnet. Themagnetic force transfer unit is for example: to move the magnetic sourcein the circumferential lateral surface direction of the packing unit orthe vertical direction (including oscillation; the same applieshereunder); to move the magnetic source enabling it to come closer or goaway with respect to the packing unit, or to vertically move, rotate, orhorizontally move the packing unit relatively with respect to themagnetic source. Combinations of modification of the magnetic force andthe movement of the position of the magnetic source allows varioustreatments to be performed on the magnetic supports.

According to the second aspect of the present invention, by providing amagnetic source which can modify the relative position or velocitybetween the packing unit or the mixture solution, and the magneticsource, or the magnetic force itself the magnetic support and the hostare controlled to move relatively therebetween, so as to increase thenumber of collision or the rate of collision between the magneticsupports and the host, to densify the magnetic support, or to combinethem both, enabling promotion of the introduction of the biologicalmaterial. At this time, the magnetic forces of the magnetic sources areapplied from at least two outside directions with the packing unittherebetween so that the combined magnetic force applied to the magneticsupport counteract each other to avoid being unidirectionally biasedagainst gravity, and the time is extended until the magnetic supportsare attached to the wall. In this case, for example, when magneticforces having similar strengths are horizontally applied in the facingdirections with the packing unit therebetween, the magnetic supportlayer can be obtained. Furthermore, by changing the position of themagnetic source or the packing unit along the circumferential direction,the magnetic support layer is extended evenly. If the layer isvertically moved in this state, the number of collisions, the rate ofcollisions, or the chance of encounters can be increased.

A third aspect of the present invention is an apparatus for introducinga biological material wherein the introduction treatment unit moves themagnetic supports and the host relatively to each other in a state wherea large number of the magnetic supports in the solution contained in thepacking unit are developed by the magnetic force.

Here, “a state where a large number of magnetic supports are developedby the magnetic force the solution” means a state where a large numberof the magnetic supports are spread and distributed in a certain area inthe solution by receiving the magnetic force. This developed stateincludes a planar case and a three dimensional case, and is determinedaccording to the distribution state of the host. The magnetic supportsin the developed state are in suspension unless the magnetic force ismoved. The magnetic support can be readily moved by moving the magneticforce.

For example, in the state where the host is widely scattered in thesolution, the large number of magnetic supports are preferably developedin a planar form for being densified more than the surroundings so as topartition the packing uric and are controlled so as to move them in thenormal direction of the developed surface. Moreover, if the host isaggregated in a partial area, the large number of magnetic supports aredeveloped in the solution so as to have the cross-section covering thearea, and are moved towards the direction of the area in the normaldirection of the developed surface. In this manner, in a state where thelarge number of the magnetic supports are developed in the solutionaccording to the distribution state of the host, the magnetic supportsare moved in a direction to increase the chance of collision orencounter with the host along the normal direction of the developedsurface of the magnetic supports.

Here, in order to realize the “developed state by the magnetic force”,it is necessary to apply the magnetic force from at least two directionswith the packing unit therebetween.

The third aspect of the present invention is to increase the number ofcollision or the rate of collision, or to increase the chance ofencounter of the magnetic support, by making relative movement betweenthe magnetic supports and the host in a state where the magneticsupports are developed in the solution in the packing unit. Therefore,attachment due to the collision with the packing unit is avoided, andthe magnetic support layer is formed. Hence the number of collisions,the rate of collisions, or the chance of encounter with the magneticsupport can be increased more widely.

A fourth aspect of the present invention is an apparatus for introducinga biological material wherein the magnetic support is a particle havinga major axis and a size allowing entry into the host along a major axisdirection.

Here, the magnetic support is preferably magnetized or magnetizablealong the major axis direction. Accordingly the magnetic support can bereadily controlled since it can be moved along the direction of themagnetic force. Moreover, if the magnetic support has a rotationallysymmetric axis along the major axis direction, it can be readilycontrolled, since it can travel accurately in the intended direction byspinning it according to how the magnetic support travels.

In the fourth aspect of the present invention, the magnetic support hasa major axis longer than the other axis and a size allowing entry intothe host along the major axis direction, and can carry the biologicalmaterial. Therefore it readily enters the host.

A fifth aspect of the present invention is an apparatus for introducinga biological material wherein an introduction adjuvant for helping tointroduce the biological material into the host is contained in thepacking unit together with the biological material. Here, “introductionadjuvant” includes for example: a calcium chloride which increases thecell membrane fluidity when a bacterial gene is introduced so as tofacilitate the taking in of the plasmid, PEG (polyethylene glycol) whichprotoplastizes the animal cell membrane; chitinase which dissolves thecell wall; and an aggregation accelerant which promotes the aggregationof the hosts themselves since the introduction is promoted by increasingthe chance of contact with the host. The aggregation accelerant includesfor example a bivalent metal ion and the like which forms crosslinksbetween cells.

In the fifth aspect of the present invention, the introduction adjuvantfor helping to introduce the biological material into the host iscontained in the packing unit together with the biological material.Accordingly, by facilitating the host to take in the biologicalmaterial, or by increasing the chance of contact of the magnetic supportwith the host, the introduction efficiency can be increased.

A sixth aspect to the present invention is an apparatus for introducinga biological material wherein the magnetic support has a carrier forcaning the biological material.

Here, the carrier expands the area to contact with the biologicalmaterial or the introduction adjuvant, enabling it to carry thebiological material in a readily separable manner when the magneticsupport enters the host, or comes into contact with or is adhered to thehost by physical adsorption, electrical interaction, or the like. Forexample, it includes concavities, perforations, or gaps provided in themagnetic support itself, or other material or a support which is fixedor bonded to the magnetic support. An example of the concavitiesincludes an annular groove provided along the circumference of theapproximate center on the side face of the magnetic support. An exampleof perforations includes a magnetic material carrying a porous materialby coating or the like if the magnetic material itself is processed, ora porous gel materials such as a cellulose gel, hydroxy-apatite, and thelike caning the magnetic material and/or the biological material. Thecarrier may be provided inside of the magnetic support.

An example of other material or a support which is fixed or bonded tothe magnetic support includes one or more filamentary supports. Thebiological material is carried by the filamentary supports. Thefilamentary supports are used, for example in a case where the host isnot strong, such as a case where the cell membrane is weak, requiringintroduction under a mild condition, in a case of a dilute fungusliquid, or in a case where handling is facilitated if they areaggregated. In this case, the respective filamentary supports are notintroduced into the host, but a load is applied to the hosts entwinedwith the filamentary supports by controlling by a magnet, so that theforce is applied as a line, and not as a point, to reduce damage. Sincethis case does not require processing of the magnetic support directly,manufacture is facilitated.

Moreover, a composite particle having a plurality of magnetic particlesconnected may be used as the magnetic support. A gap or necking of thejunction between magnetic particles may be used as the carrier.

In the sixth aspect of the present invention, by providing the carrierfor the magnetic support, the capacity of carrying the biologicalmaterial and the like can be increased and the introduction can bereliably performed.

A seventh aspect of the present invention is an apparatus forintroducing a biological material wherein in the magnetic support, bothends or one end along the major axis are formed in a tapered shape.Accordingly, the magnetic support can readily enter the host. In theseventh aspect of the present invention, the opposite ends or one endalong the major axis of the magnetic support are formed in the taperedshape. Therefore, it is possible to facilitate entry into the host suchas a cell having a hard cell wall or a cell membrane which is notsurface treated by the introduction adjuvant, and to enter reliably.

An eighth aspect of the present invention is an apparatus forintroducing a biological material wherein the introduction treatmentunit performs introduction control based on the properties, the amount,or the density of the host, the biological material, or the magneticsupport.

Here, the properties include physical properties, biological properties,and chemical properties. The physical properties include for example,strength, size, shape, mass, susceptibility, and the like. Thebiological properties include environmental conditions such as optimumtemperature and the like. The chemical properties include properties ofthe host or the biological material with respect to acid or alkali, andtoxicity with respect to metals, and the like. Whether or not themagnetic support readily enters by surface treatment, is included in thephysical properties. For example, the momentum of the magnetic supportto collide with the host is reduced with respect to a host having lowstrength, so as to avoid breaking down the host due to collision orencounter with each other. Moreover, with respect to a host havingstrength such as a plant cell with a cell wall, the momentum of themagnetic support to be collided with the host is increased to apply aforce to pierce the cell wall. These data on the properties, the amount,and the density are input into an input device provided for theapparatus, so as to process them through an information processor whichis connected to the apparatus thr introducing a biological material, andthereby control the magnetic force including the optimum magneticsource.

In the eighth aspect of the present invention, since the introduction iscontrolled based on the properties, the amount, or the density of thehost, the biological material, or the magnetic support, introductiontreatment having variety and generality can be performed in an optimumway for various hosts and various biological materials.

A ninth aspect of the present invention is an apparatus for introducinga biological material wherein the packing unit has a liquid passagethrough which the mixture solution can pass, and has a pressure adjusterwhich draws and discharges the solution by adjusting the pressure in theliquid passage, as the magnetic force control unit.

Here, a storage unit which can stole, the solution may be provided onthe packing unit in addition to the liquid passage. The liquid passageor the whole of the packing unit may be detachably attached to thenozzle of the pressure adjuster.

In the apparatus for introducing a biological material according to theninth aspect of the present invention, a liquid passage through whichthe mixture solution can pass, is provided in the packing unit.Therefore, by drawing and discharging the solution, the mixture solutioncan be readily put into the packing unit or discharged. Moreover, bycausing various relative motions between the host contained in themixture solution which is mixed by the drawing and discharging motion,and the magnetic support which is moved by the magnetic source, thechance of collision or encounter can be increased.

A tenth aspect of the present invention is an apparatus for introducinga biological material provided with a transfer mechanism which enablesrelative movement between the packing unit and the position of theintroduction treatment where the introduction treatment unit can performthe introduction treatment with respect to the packing unit. Here, theposition of the introduction treatment is for example a position wherethe magnetic source is close thereto. The movement by the transfermechanism, and the movement of the magnetic source or the packing unitfor the introduction treatment by the introduction treatment unit aregenerally different, but may be performed using a common mechanism.

In the tenth aspect of the present invention, the transfer mechanismwhich enables relative movement of the packing unit between thepositions of the introduction treatment where the introduction treatmentunit can perform the introduction treatment with respect to the packingunit is provided. Therefore, it is not necessary to provide an apparatusrequired to perform the introduction treatment, for example the magneticsource, close to the respective packing units. It is sufficient to movethe packing unit to the position where the apparatus requited to performthe introduction treatment, for example the magnetic source, is used,only when performing the introduction treatment. Therefore, it issufficient to provide a small number of positions of the introductiontreatment for a large number of packing units, thus enablingsimplification of the apparatus structure and a reduction inmanufacturing cost. Moreover, since it is not necessary to provide themagnetic source close to the respective packing units, the apparatusstructure can be integrated and manufactured in a compact size.Furthermore, by preparing the positions of the introduction treatment soas to have a magnetic source which applies various magnetic forcesaccording to purpose, various introduction treatments can be performedfor one packing unit, and hence the variety and generality are high.Moreover, various treatments including the introduction treatment can bestandardized and automatically performed using the packing unit.

An eleventh aspect of the present invention is an apparatus forintroducing a biological material wherein the magnetic source comprisesa plurality of electromagnets provided around the packing unit, and themagnetic force control unit electrically modifies the magnitude of themagnetic force of the electromagnets. The magnetic support is movedtoward the larger magnetic force until the magnitude of the magneticforce is modified at a certain distance, and then it is moved in theopposite direction. By controlling to repeat this process, the chance ofcollision or encounter of the magnetic support with the host can bepromoted. In this case, if the respective pair of electromagnets of themagnetic source are arranged to face each other around the packing unitwith the packing unit therebetween for example, and are controlled so asto switch the magnetic force, the magnetic support can be readilyreciprocated in the solution and the magnetic supports can be movedaround for a long time extensively.

In the eleventh aspect of the present invention, since the plurality ofelectromagnets serving as the magnetic source are arranged around thepacking unit and the introduction treatment is performed only bycontrolling the magnitude of the magnetic force, it is not necessary toprovide a mechanically movable part, thus simplifying the structure andelongating the life of the apparatus.

A twelfth aspect of the present invention is an apparatus forintroducing a biological material having a plurality of permanent magnetblocks or electromagnets serving as the magnetic source which aremovably provided around the packing unit, and the magnetic force controlunit moves the magnetic sources with respect to the packing unit. Themagnetic sources are moved for example, to come closer, away, verticallyor in the circumferential direction with respect to the packing unit. Atthis time, by providing the respective pairs to face each other with thepacking units therebetween and making the magnetic source closer andapart, periodic movement such as reciprocation become readily possibleso that the magnetic supports can be moved around for a long timeextensively.

In the twelfth aspect of the present invention, the plurality ofpermanent magnet blocks or electromagnets serving as the magnetic sourceare movably provided around the packing unit, and the magnetic source ismade closer and apart, so that various complex movements such asoscillation, accelerated motion, or the like can be produced.

A thirteenth aspect of the present invention is an apparatus forintroducing a biological material wherein the magnetic source is anannular magnet having predetermined magnetic poles which are arrangedaround the packing unit in a circular tubular shape, and the magneticforce control unit has: a magnetic source transfer unit which enablesmovement of the magnetic source along the radial direction, axialdirection, and the circumferential direction of the packing unit or apacking unit transfer unit which enables movement of the packing unit;or a mixture solution transfer unit which moves the mixture solution.

The “mixture solution transfer unit which moves its mixture solution” isto move the mixture solution within the packing unit. If there is aliquid passage, it corresponds to the pressure adjuster which can drawand discharge the mixture solution. The axis of the annular magnet ispreferably movable approximately coaxially in parallel, or inclined withrespect to the axis of the packing unit. The magnetic poles arepreferably distributed approximately evenly in as wide a range aspossible along the torus so that the magnetic forces can be applied firmmany directions as the “predetermined magnetic poles”. Accordingly, themagnetic supports can be distributed to divide the packing unit intoupper and lower parts in a layered planar form. Therefore, by moving thelayer vertically, the chance of widely encountering with the host in thesolution is further increased.

In the thirteenth aspect of the present invention, the annular magnetaround the packing unit is used so as to readily apply the magneticforces to the packing unit from many directions, so that the magneticsupports are developed in higher density in a planar shape than in otherareas so as to approximately partition the packing unit inside thepacking unit and be moved in a predetermined direction, thus increasingthe chance of collision or encounter of the magnetic support with thehost. Moreover since the number of magnets is only one, the apparatusstructure or the support structure is simplified.

A fourteenth aspect of the present invention is an apparatus forintroducing a biological material wherein a plurality of the packingunits are arranged along a horizontal line, and the introductiontreatment unit has: two linear magnetic sources provided with thepacking unit therebetween along the horizontal line, and having magneticpoles provided respectively in positions corresponding to the respectivepacking units; and a magnetic force control unit which enables relativemovement between the magnetic supports and the host by changing therelative position between the packing unit or the mixture solution andthe linear magnetic sources, or changing the magnetic force itself dueto the linear magnetic sources.

Here, “changing the relative position between the packing unit or themixture solution and the linear magnetic sources” means for example, tomove the linear magnetic sources closer, away, vertically, orhorizontally with respect to the packing unit or the mixture solution.Alternatively, it means to move the packing unit closer, away orvertically or horizontally with respect to the linear magnetic sources,to rotate the respective packing units about their axes, or to move themixture solution with respect to the packing unit. Accordingly thedirection of the magnetic fortes of the linear magnetic sources can becontrolled to apply from various directions, so that the number ofcollisions or the rate of collision or the chance of encounter can beincreased.

In the fourteenth aspect of the present invention, a plurality ofpacking units are arranged in parallel in a horizontal line, and themagnetic sources in the horizontal line are arranged with the packingunits therebetween, along the lines of the packing units. Therefore, theapparatus structure can be simplified and the introduction treatment canbe integrally performed, so that the introduction treatment can befurther effectively performed.

A fifteenth aspect of the present invention is an apparatus forintroducing a biological material wherein the linear magnetic source hastwo linear support bodies provided on opposite sides with the arrayedpacking units therebetween, along the horizontal line, and a pluralityof permanent magnets or electromagnets arranged at intervals andpositions corresponding to the respective packing units, in the linearsupport bodies.

The permanent magnet or the electromagnet may be formed by bending intosemiannular shapes around half portions of the packing unit respectivelyso as to apply the magnetic threes from many directions.

In the fifteenth aspect of the present invention, the linear magneticsources are arranged at intervals and positions corresponding, to thepacking units arranged in the linear support bodies. Therefore, thepermanent magnets or the electromagnets are arranged for each of thepacking units, thus simplifying the structure and facilitating thetreatment.

A sixteenth aspect of the present invention is an apparatus forintroducing a biological material wherein each of the packing units hasa liquid passage through which a mixture solution can pass, and apressure adjuster which draws and discharges the solution by adjustingthe pressure in the liquid passage.

In the sixteenth aspect of the present invention, each packing unit hasa liquid passage through which the mixture solution can pass. Therefore,the drawing and discharging of the solution enables relative movementbetween the host and the magnetic support, increasing the number ofcollisions or the rate of collisions, or increasing the chance ofencounter.

A seventeenth aspect of the present invention is an apparatus forintroducing a biological material wherein the introduction treatmentunit has a magnetic separation unit whereby hosts having the magneticsupport introduced thereinto or adhered thereto, are attached to theinner wall of the packing unit and separated from the mixture solutionin the packing unit, by controlling the magnetic force applied to theinside of the packing unit.

In order to separate the unused magnetic supports which have not enteredinto or been adhered to the host, from in the mixture separated by themagnetic force of the magnetic separation unit, it is preferably that afilter having a pore size between the host size and the magnetic supportsize is provided to partition a predetermined part in the liquidpassage, or that a holder having the filter is detachably attached tothe tip end of the liquid passage. By doing this, after introductiontreatment, the solution suspended with only the mixture separated by theattachment to the inner wall of the packing unit by the magnetic forceis drawn and discharged with respect to the packing unit through thefilter, so that only the host having the magnetic support enteredthereinto or adhered thereto is extracted in the filter, and the unusedmagnetic supports are removed. Alternatively, the situation may be suchthat, after introduction treatment, before the separation by themagnetic forte, the mixture solution is drawn and discharged withrespect to the packing unit through the filter, so that the unusedmagnetic supports are removed, and then the separation is performed bythe magnetic force so as to extract the host having the magnetic supportentered thereinto or adhered thereto.

The liquid passage may be a tip detachably provided on the nozzle of thepressure adjuster. The filter may be provided on the tip. Moreover, inorder to remove various other impurities except for the target host, oneor more filters having different pore sizes may be provided in theliquid passage or the holder. Furthermore, a plurality of holdersprovided with the filters may be detachable in multi-stages with respectto the liquid passage. Accordingly, only the host having the magneticsupport entered thereinto or adhered thereto, that is the host havingthe target biological material introduced can be separated, so thatunnecessary substances such as the unused magnetic supports, impurities,and the like are removed, so that reliable introduction of thebiological material can be performed.

In the seventeenth aspect of the present invention, regarding themagnetic separation unit by which the hosts having the magnetic supportintroduced thereinto or adhered thereto are attached to the inner wallof the packing unit to be separated by the magnetic force, the magneticsource which is used for increasing the chance of collision or encounterof the magnetic support with the host may be used, so that the structurecan be simplified and the separation can be efficiently and quicklyperformed.

An eighteenth aspect of the present invention is an apparatus forintroducing a biological material wherein then magnetic separation unithas a separation instruction unit which instructs the magnetic forcetransfer control unit to apply only a unidirectional magnetic fametowards the wall of the packing unit.

Accordingly, the magnetic support collides with the inner wall of thepacking unit along the above direction, and is attached thereto.

In the eighteenth aspect of the present invention, it is sufficient ifthe magnetic separation unit merely instructs the magnetic source toapply only the unidirectional magnetic force towards the wall of thepacking unit. Hence the structure can be simplified and themanufacturing cost can be reduced.

A nineteenth aspect of the present invention is a method of introducinga biological material comprising: a mixing step wherein a large numberof magnetic supports carrying a biological material introduced into ahost such as a cell, and a large number of the hosts are mixed in asolution to make a mixture solution, which is then put into one or morepacking units; and an introduction treatment step wherein the magneticsupports are moved relatively with respect to the host by controlling amagnetic force applied to the packing unit so as to introduce thebiological material into the host.

In the present invention, by moving the magnetic supports around withrespect to the host in the solution in the packing unit by the magneticforce, the chance of collision or encounter with the host suspended orcontained in the solution can be increased. Accordingly, theintroduction of the biological material into the host is promoted. Atthis time, if the magnetic force is applied from at least two directionswith the packing therebetween, the movement of the magnetic supports isnot finished by being attached to the packing unit in the solution onlyby a unidirectional motion by the magnetic force applied, but can becontinued in the solution for a long time by releasing the attachmentstate, or by initially avoiding the attachment state.

According to the nineteenth aspect of the present invention, similareffects to those described in the first aspect of the present inventionare demonstrated.

A twentieth aspect of the present invention is a method of introducing abiological material wherein in the introduction treatment step, themagnetic supports and the host are moved relatively to each other bychanging the relative position or the velocity between the packing unitor the mixture solution and the magnetic field, or the magnetic forceitself.

Here, by applying the magnetic force from at least two directions withthe packing unit therebetween, from the outside of the packing unit intothe packing unit, the magnetic supports are moved around in the solutionavoiding a situation such as where the magnetic supports are attachedonto the packing unit or precipitated at the bottom of the packing unitby a unidirectional magnetic force, so that the chance of collision orencounter with the host can be increased and the introduction of thebiological material into the host can be promoted.

According to the twentieth aspect of the present invention, similareffects to those described in the second aspect of the present inventionare demonstrated.

A twenty-first aspect of the present invention is a method ofintroducing a biological material wherein in the introduction treatmentstep, the magnetic supports and the host are moved relatively to eachother in a state where a large number of the magnetic supports in thesolution contained in the packing unit are developed solution by themagnetic force.

According to the twenty-first aspect of the present invention, similareffects to those described in the third aspect of the present inventiondemonstrated.

A twenty-second aspect to the present invention is a method ofintroducing a biological material wherein, the introduction treatmentstep uses a magnetic support being a particle having one major axis, andhaving a size allowing entry into a host along the major axis direction,and moves the magnetic support to enter the host so as to introduce thebiological material. Here, the magnetic support is magnetized or formedto be easily magnetizable, in the major axis direction, so as to enablethe magnetic support to be moved in the major axis direction, thusfacilitating the entry using the magnetic force.

According to the twenty-second aspect of the present invention, similareffects to those described in the fourth aspect of the present inventionare demonstrated.

A twenty-third aspect of the present invention is a method ofintroducing a biological material comprising a separation step after theintroduction treatment step wherein the host having the magnetic supportentered thereinto or adhered thereto is attached to the inner wall ofthe packing unit and separated, by controlling the magnetic force insideof the packing unit.

After the separation step, a removal step wherein unused magneticsupports which did not enter into or were not adhered to the host andthe like are removed from among the substances attached to the inner allof the packing unit. The removal step is performed for example, by usinga filter provided so as to partition the liquid passage provided in thepacking unit, or a holder having a filter detachably attached to theliquid passage, and drawing and discharging a solution with respect tothe packing unit and the like through the filter. Moreover, a removalstep may be provided for removing one or more other impurities exceptfor the magnetic support using one or more types of filter. Accordingly,an even more reliable introduction treatment can be performed.

Furthermore, according to the twenty-third aspect of the presentinvention, similar effects to those described in the seventeenth aspectof the present invention are demonstrated.

A twenty-fourth aspect of the present invention is a method ofintroducing a biological material wherein the mixing step mixes themagnetic support and the biological material to be introduced into thehost in solution, so that the biological material is carried in themagnetic support.

According to the twenty-fourth aspect of the present invention, bymixing the magnetic support and the biological material to be introducedinto the host in solution, the biological material can be readilycarried, making it suitable for automating the treatment.

A twenty-fourth aspect of the present invention is a method ofintroducing a biological material comprising a culturing step whereinafter the separation step, in a state where the separated hosts havingthe magnetic support adhered thereto or entered thereinto are pooled inthe packing unit, the hosts are relatively moved to inside a containercontaining a medium and the hosts are pooled in the container andcultured.

According to the twenty-fifth aspect of the present invention, in thestate where the host having the magnetic support entered thereinto oradhered thereto are separated by using the magnetic force and attachedto the inner wall of the packing unit, the host can be relatively movedto inside into the container containing the medium and put into themedium, enabling the culturing treatment to be performed automatically.Therefore the treatment can be standardized and automatically performed.

A twenty-sixth aspect of the present invention is a method ofintroducing a biological material wherein in the culturing step, thehosts having the magnetic support entered thereinto or adhered theretoare separated and removed from the cultured hosts by applying a magneticforce, so as to exclusively obtain purely cultured hosts.

According to the twenty-sixth aspect of the present invention, the hosthaving the magnetic support entered thereinto or adhered thereto, andwhich has initially been placed into the medium in the culturing step,can be cultured while initially being separated from the cultured hostby applying the magnetic force. Therefore, the host having the magneticsupport entered thereinto or adhered thereto can be readily removed.Accordingly, a purely cultured cultured host can be exclusively obtainedeasily and reliably. Therefore, the negative affect to the organism dueto the presence of the magnetic support in the cell can be removed.

A twenty-seventh aspect of the present invention is a method ofintroducing a biological material comprising: an introduction step formaking magnetic supports carrying the biological material to beintroduced into the host collide or encounter with the host using amagnetic force so as to introduce the biological material into the host;a separation step for separating the host having the magnetic supportentered thereinto or adhered thereto; a culturing step for culturing thehost using the separated host; and an extraction step for extracting apurely cultured host by separating the host which initially has themagnetic support entered thereinto or adhered thereto, from inside thecultured host. Here, the separation step preferably includes animpurities removal step for removing unused magnetic supports which werenot adhered to or did not enter into the host, and foreign matter orimpurities using a filter.

According to the twenty-seventh aspect of the present invention, thetreatment from the introduction of the biological material into thehost, to the culture of the host having the biological materialintroduced thereinto can be automatically performed and standardized.

A twenty-eighth aspect of the present invention is a magnetic supportfor introducing a biological material, which is a magnetic support in aparticle shape having one major axis, and capable of carrying biologicalmaterial to be introduced into a host such as a cell, and having a sizeallowing entry into the host along the major axis direction. Here, themagnetic support is preferably magnetized or formed so as to be easilymagnetized, along the major axis direction, and is formed so as to bereadily moved along the major axis direction under control. Moreover,the magnetic support preferably has an axial symmetry in relation to themajor axis.

According to the twenty-eighth aspect of the present invention, similareffects to those described in the fourth aspect of the present inventionare demonstrated.

A twenty-ninth aspect to the present invention is a magnetic support forintroducing a biological material wherein the magnetic support has acarrier for carrying the biological material. Here, the carrier is asdescribed in the sixth aspect to the present invention.

According to the twenty-ninth aspect of the present invention, similareffects to those described in the sixth aspect of the present inventionare demonstrated.

A thirtieth aspect of the present invention is a magnetic support forintroducing a biological material wherein in the magnetic support, bothends or one end along a major axis are formed in a tapered shape. Thedegree of tapering is as described in the seventh aspect of the presentinvention.

Furthermore, according to the thirtieth aspect of the present invention,similar effects to those described in the seventh aspect of the presentinvention are demonstrated.

The thirty-first aspect of the present invention is an apparatus forintroducing a biological material comprising; one or more packing unitscontaining a mixture solution having a large number of magnetic supportscarrying a biological material to be introduced into a host such as acell, and a large number of the hosts in a solution; an introductiontreatment unit which moves the magnetic supports relatively with tweetto the hosts in the solution by controlling a magnetic force applied toinside the packing unit so as to introduce the biological material intothe host; and a perforation treatment unit which perforates the host.

Here, “perforate the host” means to make holes in a boundary region incontact with the outside of the host so as to facilitate entry of themagnetic support on the outside into the host, or facilitateintroduction of the biological material into the host. The perforationtreatment is normally performed on the host in the packing unit, howeverit is not limited to this. If the host is a cell, the boundary is a cellmembrane or a cell wall. In order to make holes in the boundary of thehost, for example a perforation force is applied to the host byelectrical discharge or ultrasound cavitation “Perforation force” meansa certain force for making holes on the boundary of the host. In thecase of electrical discharge, if the host is a cell, for example, a highvoltage of about several thousands of volts/cm is applied at severaldozens of microsecond pulses. Moreover, in the case where the magnitudeof the voltage is modified, the pulse width may be modified accordingthereto. If the other conditions are the same, then in the case of lowvoltage, the pulse width is elongated more than for the case of a highvoltage. In the case of high voltage, the pulse width is shortened morethan for the case of a low voltage. Accordingly, small holes are made onthe boundary of the host, for example the cell membrane for a shorttime, facilitating making the magnetic support enter into or beintroduced to the biological material. According to the presentinvention, even if the magnetic supports are not collided with the hostthe entry efficiency of the magnetic support or the introductionefficiency of the biological material to the host may be increased byincreasing the chance of encounters of the magnetic support and thehost. Therefore, control is facilitated. According to the presentinvention, the objective of the electroporation for perforating the cellmembrane to introduce only the gene on the outside, is expanded togeneral hosts, and the boundary of the host on the outside is perforatedso that the magnetic support can enter the host.

A thirty-second aspect of the present invention is an apparatus forintroducing a biological material wherein the introduction treatmentunit comprises: a magnetic source which can apply a magnetic force tothe inside of the packing unit; and a magnetic force control unit whichcontrols relative movement between the magnetic supports and the host bychanging the relative position or the velocity between the packing unitor mixture solution and the magnetic field, or the magnetic force itselfdue to the magnetic source.

According to the thirty-second aspect of the present invention, similareffects to those described in the second aspect of the present inventionare demonstrated.

A thirty-third aspect of the present invention is an apparatus forintroducing a biological material wherein the perforation treatment unithas: a perforation force source which can apply a perforation force byan electric field, or ultrasound, or the like; and a perforation forcesource control unit which controls the perforation force source.

The perforation force source is preferably provided within the packingunit. Accordingly, the perforation three can be reliably and efficientlyapplied to the host in the packing unit.

If the packing unit has a liquid passage through which the mixturesolution can pass and the magnetic force control unit has a pressureadjuster which draws and discharges the solution by adjusting thepressure within the liquid passage, the perforation treatment unit isprovided with a perforation force source so as to perform theperforation treatment on the host passing through the liquid passage.

Accordingly, since the treatment by the perforation treatment unit isperformed in the liquid passage, the perforation treatment can beapplied efficiently to the host passing through the liquid passage.

Moreover, if the packing unit has a storage unit which stores a mixturesolution, the perforation treatment unit may be provided with aperforation force source so as to perform perforation treatment on thehost in the mixture solution stored in the storage unit. Accordingly,since in this case the perforation treatment is performed at theposition where the introduction treatment is performed, the entrance ofthe magnetic support into the perforated host is further readilycontrolled.

The perforation force source is not necessarily provided on the packingunit, but may be provided in an external container for containing thesolution to be supplied to the packing unit. Accordingly, the perforatedhost can be drawn into the packing unit.

The control of the perforation force tree includes the control of thetype, position, the size, the direction, or the time of the perforationforce.

If electrical discharge is used as the perforation force, theperforation force source is a pair of facing electrodes. If ultrasoundis used as the perforation force, the perforation force source is anultrasonic oscillator. The selection of the voltage to be applied to theelectrode, and the selection of positive electrodes and negativeelectrodes may be changed according to the size, the position, or theoperating direction of the biological material to be introduced, thehost, or the magnetic support, or according to the position or thecontrol of the magnetic source, or according to the time. Accordingly,the entrance of the magnetic support or the introduction of thebiological material can be further efficiently performed. Moreover, thearrangement may be such that both of electrical discharge and ultrasoundas the perforation force source are applied to the host in the packingunit. For that purpose, a plurality of types of perforation forcesources are provided.

A thirty-fourth aspect of the present invention is an apparatus forintroducing a biological material wherein the perforation force sourcecontrol unit controls the perforation force source based on theproperties, the amount, or the density of the host, the biologicalmaterial, or the magnetic support.

Accordingly, by applying the optimum perforation force to the host orthe like being used, the biological material can be reliably andefficiently introduced.

Here, “properties” includes the physical properties such as the size,the hardness, and the shape, and the chemical properties such asmolecular structure, repairability, and the like.

A thirty-fifth aspect of the present invention is an apparatus forintroducing a biological material wherein the perforation force sourceunit or the magnetic force control unit control the introductiontreatment and the perforation treatment so as to be executed in spatialor time association with each other.

The reason for this is that the perforation provided in the host by theperforation treatment unit may be repaired in a short time, thusrequiring consideration of the respective positions and the limes toperform the perforation treatment and the introduction treatment.

Moreover, if the magnetic three is controlled to match the direction ofthe perforation treatment of the host so as to move the magnetic supportin the direction of the host, the magnetic support readily enter thehost.

A thirty-sixth aspect of the present invention is an apparatus forintroducing a biological material wherein the packing unit has a liquidpassage through which the mixture solution can pass, and has a pressureadjuster which draws and discharges the solution by adjusting thepressure in the liquid passage, as the magnetic force control unit.According to the thirty-sixth aspect of the present invention, similareffects to those described in the ninth aspect of the present inventionare demonstrated.

A thirty-seventh aspect of the present invention is a method ofintroducing a biological material comprising: a mixing step wherein alarge number of magnetic supports carrying a biological material to beintroduced into a host such as a cell, and a large number of the hostsare mixed in a solution to make a mixture solution, which is then putinto one or more packing units; a perforation treatment step whichperforates the host such as a cell; and an introduction treatment stepwherein the magnetic supports are moved relatively with respect to thehost by controlling a magnetic force applied to the packing unit, so asto introduce the biological material into the host.

Here, the perforation treatment step is not necessarily performed afterthe mixing step, and may be performed in an external container beforethe mixing step, so as to mix the perforated host.

Moreover, if the perforation treatment step comes after the mixing step,the perforation treatment is performed within the packing unit.

According to the thirty-seventh aspect of present invention, even if themagnetic supports are not collided with the host, the entry efficiencyof the magnetic support or the introduction efficiency of the biologicalmaterial to the host, may be increased by increasing the chance ofencounter of the magnetic support and the host. Therefore, the controlis facilitated.

A thirty-eighth aspect of the present invention is a method ofintroducing a biological material wherein the perforation treatment stepis performed by applying a perforation force such as an electric field,ultrasound, or the like.

According to the thirty-eighth aspect of the present invention, similareffects to those described in the thirty-third aspect of the presentinvention are demonstrated.

A thirty-ninth aspect of the present invention is a method ofintroducing a biological material wherein the perforation treatment stepapplies the perforation force based on the properties, the amount, orthe density of the host, the biological material, or the magneticsupport.

According to the thirty-ninth aspect of the present invention, similareffects to those described in the thirty-fourth aspect of the presentinvention are demonstrated.

A fortieth aspect of the present invention is a method of introducing abiological material wherein the perforation treatment step and theintroduction treatment step are executed in spatial or time associationwith each other.

According to the fortieth aspect of the present invention, similareffects to those described in the thirty-fifth aspect of the presentinvention are demonstrated.

The forty-first aspect of the present invention is an apparatus forintroducing a biological material which further comprises: a transferdevice which separates and transfers a solution containing a host whichhas been treated by the introduction treatment unit, and has themagnetic support adhered thereto or entered thereinto, or a host havingthe magnetic support adhered thereto or entered thereinto; a containercontaining a medium; and a separation unit for separating the magneticsupport contained in the container and the host having the magneticsupport adhered thereto or entered thereinto.

Here, the separation unit may be for example, a magnetic force unitcomprising a permanent magnet or an electromagnet provided at the bottomor the lateral side of a container formed of a resin or the like whichdoes not shield the magnetic field; or may be a dispenser which has aliquid passage, a pressure adjustment device which adjust the pressurewithin the liquid passage, and a magnetic force device which can applythe magnetic force from the outside of the liquid passage into theliquid passage, with a transfer function. In the case of a containercomprising a plurality of holes, the permanent magnet or theelectromagnet is preferably provided for each of the respective holes.

According to the forty-first aspect of the present invention, byapplying the magnetic force to the container containing the medium, thehost having the magnetic support altered thereto or entered thereintocan be separated, so that the purely cultured host with a simplestructure can be easily obtained. Hence safe and reliable introductiontreatment can be performed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 show a main part of an apparatus for introducing a biologicalmaterial according to a first embodiment of the present invention.

FIG. 2 is an explanatory diagram of the operation of the apparatus forintroducing a biological material according to the first embodiment ofthe present invention.

FIG. 3 is a plan view and an elevational view of a biological materialintroduction system according to a second embodiment of the presentinvention.

FIG. 4 shows the apparatus for introducing a biological materialaccording to the second embodiment of the present invention.

FIG. 5 is a plan view showing another example of an apparatus forintroducing a biological material according to the second embodiment ofthe present invention.

FIG. 6 shows a nozzle head according to the second embodiment of thepresent invention.

FIG. 7 is an explanatory diagram of the operation of the apparatus forintroducing a biological material according to the second embodiment ofthe present invention.

FIG. 8 is an explanatory diagram of the operation of the apparatus forintroducing a biological material according to the second embodiment ofthe present invention.

FIG. 9 is an explanatory diagram of the operation of the apparatus forintroducing a biological material according to the second embodiment ofthe present invention.

FIG. 10 is an enlarged diagram of magnetic supports according to a thirdembodiment of the present invention.

FIG. 11 is a flowchart showing an example of gene therapy according to afourth embodiment of the present invention.

FIG. 12 is a flowchart showing an example of protein synthesis accordingto a fifth embodiment of the present invention.

FIG. 13 is a cross-sectional view showing a main part of an apparatusfor introducing a biological material according to a sixth embodiment ofthe present invention.

FIG. 14 is a cross-sectional view showing another example of the mainpart of the apparatus for introducing a biological material according tothe sixth embodiment of the present invention.

FIG. 15 shows a separation unit of an apparatus for introducing abiological material according to a seventh embodiment of the presentinvention.

BEST MODE FOR CARRYING OUT THE INVENTION

An apparatus for introducing a biological material and a method ofintroducing a biological material according to embodiments of thepresent invention are described based on the drawings. The descriptionof the present embodiments should not be considered as limiting thepresent invention unless particularly specified.

FIG. 1 shows a main part of an apparatus 1 for introducing a biologicalmaterial according to a first embodiment.

The apparatus 1 for introducing a biological material has anapproximately circular tubular tip 3 which is detachably attached to anozzle 2, an annular permanent magnet 4, and a magnetic source transferunit (not shown). The tip 3 corresponds to one packing unit in which amixture solution containing a large number of magnetic supports can abiological material to be introduced into a host such as cells upon use,together with a large number of the hosts are pooled. The annularpermanent magnet 4 corresponds to a magnetic source which is movablyprovided for controlling a predetermined magnetic force so that themagnetic supports in the solution in the tip 3 are moved relativelybetween the magnetic supports and the hosts, making the magneticsupports collide with or encounter the hosts. Moreover, the magneticsource transfer unit is for moving the annular permanent magnet 4 withrespect to the tip 3.

The tip 3 has a small diameter liquid passage 5 which is for drawing anddischarging a liquid with respect to a container (not shown) providedoutside, a large diameter storage unit 6 which stores the mixturesolution containing the magnetic supports, and a hollow middle diameterunit 7 in an approximately truncated conic shape which is providedbetween the liquid passage 5 and the storage unit 6. The axis of theannular permanent magnet 4 is desirably provided in an approximatecoaxial manner, inclined, or in parallel with respect to the axis of thestorage unit 6, so that the annular permanent magnet 4 is movable in theaxial, radial, and circumferential directions, and optional combinationthereof around the storage unit 6.

Moreover, an attachment unit 8 for attaching the nozzle 2 on the top ofthe storage unit 6 is provided. The nozzle 2 is communicated with apiston via a duct (not shown) and corresponds to a pressure adjuster.

The annular permanent magnet 4 is a combination of two semiannularpermanent magnets. One semiannular permanent magnet has a north pole inan area including the center, and south poles on the opposite ends. Theother semiannular permanent magnet has a south pole in an area includingthe center, and north poles on the opposite ends. These opposite endsare connected and formed into an annular shape. Lines of magnetic forceare distributed such that the density of two portions including thecenters which face each other with the storage unit 6 of the tip 3 beingthe packing unit therebetween, is even and the highest, and the densitygets lower as it goes away from the area along the torus.

In FIG. 1, the annular permanent magnet 4 can be oscillated in thedirections shown by the arrows 9 and 10 in a solid straight line, by themagnetic force transfer unit. Moreover, the nozzle 2 and the tip 3attached thereto can be rotated by a nozzle rotation unit (not shown) ina direction shown by the arrow 11 in a solid curved line.

The arrow 12 in a broken line represents a case where an attempt is madeto separate the magnetic supports by making them attach to the wall ofthe tip 3, moving the annular permanent magnet 4 along the directionlinking the magnetic poles, so that only one magnetic pole comessufficiently close to the storage unit 6 of the tip 3. These magneticsource transfer unit, nozzle rotation unit, and pressure adjusterconstitute the magnetic force control unit, which constitutes theintroduction treatment unit together with the annular permanent magnet 4being the magnetic source.

FIG. 2 explains the operation of the apparatus 1 for introducing abiological material shown in FIG. 1.

A mixture solution containing the magnetic supports carrying thebiological material to be introduced into cells being a host drawn froman external container through the liquid passage 5 of the tip 3, and thecells being the host, are placed in the storage unit 6. At this time,the magnetic force due to the annular permanent magnet 4 affects themagnetic supports contained in the mixture solution.

Moreover, due to the annular permanent magnet 4, magnetic forces ofapproximately identical degree influence the magnetic supports mainlyfrom two facing directions with the tip 3 therebetween. Therefore thebalance of the respective magnetic supports is maintained where thecombined magnetic forces almost cancel each other, keeping the magneticsupports from being attracted unilaterally to one direction, and to notbe attached to the wall of the tip 3. The magnetic supports are alsokept from falling down due to the gravity, by the magnetic force, andthe magnetic supports are developed in a high density in a layeredplanar shape in the solution. As shown in FIG. 2( a), the magneticsupports in the mixture solution form a horizontal planar magneticsupport layer 13 in an approximate letter 8 shape as shown in FIG. 2 (b)at the height where the annular permanent magnet 4 is present.

Regarding the magnetic support layer 13 in such a developed state, theannular permanent magnet 4 is oscillated and repeatedly moved along thevertical arrow 10 by the magnetic source transfer unit (not shown) andthe nozzle 2 is rotated by the nozzle rotation unit. In consequence, themagnetic support layer 13 is oscillated in the direction of the anew 14as shown in FIG. 2( a), and the layer 13 is rotated in the tip 3 asshown by the arrow in FIG. 2 (b).

Accordingly, the magnetic support layer 13 is extended to partition thetip 3 into upper and lower parts horizontally as if it is a filter or apaper provided approximately in the middle of the tip 3. Therefore thevertical oscillation of the magnetic support layer 13 using the magneticsource transfer unit can promote a closer approach of the magneticsupports to the hosts diffused in the solution passing through themagnetic support layer 13, and can increase the number of collisions orthe rate of collision, or increase the chance of encounter.

As a result, it becomes highly possible that the hosts in the solutioncollide with or encounter the magnetic supports constituting themagnetic support layer 13, making the magnetic supports come in contactwith the hosts, making the magnetic supports adhere to the hosts, ormaking the magnetic support enter the hosts.

Next is a description of a biological material introduction system 15according to a second embodiment based on FIG. 3 to FIG. 9. The samereference symbols are used for components the same as those described inthe first embodiment, and the description thereof is omitted.

As shown in a plan view of FIG. 3 (a), the biological materialintroduction system 15 has: a stage 16 on which the biological materialintroduction treatment is performed; a set of eight nozzles unit 17having the abovementioned nozzles 2 provided for the stage 16, andarranged at intervals along a horizontal line with the respective axesthereof vertical and in parallel, with the tips 3 detachably attached tothe nozzles 2 respectively; reagent baths 18 provided with variousmediums according to the host type; tip racks 19 having unused tips 3 tobe attached to the set of eight nozzles 2 arranged in a matrix form ofeight columns plate 21 having solution packing units 20 arranged in amatrix form of 8 columns×12 rows for containing a solution forperforming the biological material introduction treatment, that is, amixture solution containing a large number of magnetic supports carryinga target biological material to be introduced into a host such as cells,and a large number of hosts, mixed in solution; a container 23comprising solution packing units 22 of 8 columns×2 rows containing DNAsolution; and a container 25 comprising solution packing units 24 of 8columns×2 rows containing a solution suspended with cells as the host.One row of the solution packing units in the container 25 is a container26 for PCR.

Furthermore, the stage 16 has; a magnetic force treatment unit 27corresponding to the introduction treatment unit, by which a magneticforce can be applied to the tips 3 attached to the set of eight nozzles2 all at once; a container 29 comprising solution packing units 28 of 8columns×2 rows for containing the introduced result; and a waste vent 30for disposing the used tips 3. Reference symbols 31, 32, and 33 denoteheated/cooled areas where a temperature control unit for heating orcooling the containers in the respective areas is provided, enablingcontrol of the temperature.

The set of eight nozzles unit 17 has the set of eight nozzles 2 and theset of eight tips 3 attached thereto. A nozzle head 77 provided withvarious mechanical sections is provided to the nozzle unit 17. Thenozzle head 77 is provided with a Z-axis driving motor 81 whichvertically moves the nozzle unit 17.

Moreover, the biological material introduction system 15 has a Y-axisconveyer 82 which can move the nozzle head 77 in the Y-axis direction,and an X-axis conveyer 83 which can move the nozzle head 77 in theX-axis direction. Therefore, the nozzle head 77 is movable horizontallyand vertically within the area on the stage 16. Reference symbol 84denotes a motor for driving the X-axis conveyer 83. As shown in thefront view of FIG. 3 (b), the motor 84 moves the nozzle head 77 in theX-axis direction by driving to rotate the belt 85. Here, the Y-axisconveyer 82, the X-axis conveyer 83, and the Z-axis driving motorcorrespond to the transfer unit.

Reference symbol 86 denotes a P-axis motor which drives the piston inthe pressure adjuster communicated with the nozzle 2 to adjust thepressure in the nozzle 2 and the tip 3. The P-axis motor 86 drives torotate a ball screw 88 via a coupling 87. A nut 89 into which the ballscrew 88 is threaded, is fixed to props 89 b and 89 c for guiding via aplate 89 a. Therefore, the ball screw 88 is vertically moved by therotation, which moves a set of eight pistons (not shown) provided on amember 90 which is provided at the bottom of the ball screw 88,vertically all at once. Reference symbol 80 denotes a part provided witha rotating mechanical section for rotating the nozzle 2 around it axis.

FIG. 4 shows the magnetic force treatment unit 27 in a state where thepoint of the tip 3 attached to the respective nozzles 2 of the nozzleunit 17 is inserted into the respective holes 96.

In order to apply the magnetic force to the inside of the tip 3, thenozzle unit 17 is moved to the magnetic force treatment unit 27 by thetransfer mechanisms 82 and 83 on the stage 16, and the tip 3 is insertedinto the magnetic force treatment unit 27.

As shown in FIG. 4, the set of eight nozzles 2, and therefore the tips3, are arranged along the horizontal line. Moreover, the magnetic forcetreatment unit 27 has two linear magnetic sources 36 and 37 which haverod-shaped supporting sections 34 provided on the opposite sides withthe tip 3 therebetween, in parallel along the horizontal line, and eightpermanent magnet blocks 35 set in positions corresponding to therespective tips 3.

Here, the magnetic poles of the facing magnet blocks 35 of therespective linear magnetic sources 36 and 37 may be the same ordifferent for the south poles and the north poles. Moreover, themagnetic poles of the respective linear magnetic sources 36 and 37 maybe arranged to be alternately different in some cases, while the samemagnetic poles may be arranged in other cases.

The respective linear magnetic sources 36 and 37 are provided so as tobe able to come closer or go away with respect to the nozzle unit 17 bya close/away direction transfer mechanism, and so as to be movable alongthe array direction of the nozzles 2 of the nozzle unit 17 by an arraydirection transfer mechanism. The close/away direction transfermechanism has: motors 91; ball screws 92 driven to rotate by the motors91; holddown members 93 which are provided so as to be threaded by theball screws 92 and fixed to the respective floor boards (not shown) ofthe array direction transfer mechanism which is movably provided withrespect to the stage 16 in the horizontal plane; two props 95 which areinserted through the holddown members 93 and connected with thesupporting sections 34; and movable members 94 which support the motors91, the ball screws, and the props 95. The supporting sections 34 can bemoved back and forth with respect to the nozzle unit 17 by driving themotors 91. The array direction transfer mechanism is a mechanism to movethe linear magnetic sources 36 and 37 separately by a predetermineddistance (about the distance between adjacent nozzles 2) in thehorizontal plane along the array direction of the nozzles 2 of thenozzle unit 17. The array direction transfer mechanism is provided withthe two floor boards which are separately provided so as to be movablealong the rail (not shown) along the array direction, provided on thestage 16, instead of the linear magnetic sources 36 and 37 of theclose/away direction transfer mechanism, and is respectively providedwith holddown members fixed to the stage 16, instead of the holddownmembers 93.

In this manner, with respect to the tip 3, the lines of magnetic forcecan be moved by moving the tip 3 itself in the Z-axis direction, thelines of magnetic force can be moved by moving the supporting sections34 freely back and forth with respect to the tip 3, and the lines ofmagnetic force can be moved by moving the supporting sections 34horizontally along the array direction of the nozzles 2. Here, thetransfer mechanism corresponds to the magnetic some transfer unit.

An example shown in FIG. 5 illustrates linear magnetic sources 99 and100 on which semiannular permanent magnets 97 and 98 are arranged atintervals corresponding to the respective tips 3. The semiannularpermanent magnets 97 and 98 respectively correspond to the twosemiannular permanent magnets of the annular permanent magnet 4described in FIG. 1.

FIG. 6 shows the rotating mechanical section 80 provided in the nozzleunit 17. The rotating mechanical section 80 has: a support plate 101; amotor 102 which is provided on the support plate 101, for rotating therespective nozzles 2 about their axes; and a timing belt 103 which isprovided above the support plate 101 and spanned around a driving pulley108 of the motor 102, nozzle pulleys 109 provided for the respectivenozzles 2, and pulleys 104, 105, and 106, to rotation chive the nozzles2. Reference symbol 107 denotes a rotary joint for rotatably connectingalong the axial direction, and the piston is connected at the top.Reference symbols 110 and 111 denote bearings.

FIG. 7 and FIG. 8 schematically show the position of the respective tips3 of the nozzle unit 17, and the linear magnetic sources 36 and 37 inthe magnetite force treatment unit 27.

FIG. 7 (1) schematically shows a case where the respective distances ofthe linear magnetic sources 36 and 37 are apart from the nozzle unit 17enough to weaken the magnetic force influencing the inside of the tip 3.In this case, as show FIG. 7 (2), no arrow is shown representing themagnetic force inside each tip 3, since the magnetic force inside eachtip 3 is weak

FIG. 7 (3) shows a case where the respective linear magnetic sources 36and 37 are made closer from the opposite sides, to the nozzle unit 17 bythe transfer unit. In this case, as shown in FIG. 7 (4), each tip 3receives the magnetic force vertically.

FIG. 7 (5) shows a case where one linear magnetic source 36 is made evencloser to the nozzle unit 17 and the other linear magnetic source 37 ismade further away from the nozzle unit 17. In this case, as shown inFIG. 7 (6), the magnetic force is applied in one direction only.Therefore, the magnetic supports contained in the tip 3 are attracted inone direction only, and attached to the internal wall of the tip 3, andthus separated.

FIG. 8 shows a case where the linear magnetic sources 36 and 37 aredisplaced by a shorter distance than the distance between the adjacenttips 3 along the horizontal line, in a state where the linear magneticsources 36 and 37 are close to the nozzle unit 17. FIG. 8 (1) shows thestate where the linear magnetic source 36 is moved to the left and thelinear magnetic source 37 is moved to the right respectively by about ahalf of the distance between the adjacent tips 3. The magnetic forceinside the respective tips 3 in this case is applied in the directionsshown in FIG. 8 (2). FIGS. 8 (3) and (4) show a case where the case ofthe FIGS. 8 (1) and (2) is displaced in reverse directions of left andright. Similarly to the present example, the magnetic forces are appliedto oppose each other with the respective tips 3 therebetween, by movingthe linear magnetic sources 36 and 37. Therefore, the magnetic supportsinside the tip 3 can be influenced so as to develop.

FIG. 9 shows a state seen from the horizontal direction along thehorizontal line, for when the tips 3 attached to the set of eightnozzles and linearly arranged, are moved to the magnetic force treatmentunit 27 and inserted into the openings. Therefore, in FIG. 9, only onetip 3 out of eight tips is illustrated, and the magnetic force treatmentunit 27 is illustrated such that the rod shaped linear magnetic sources36 and 37 provided in parallel with the set of eight nozzles 2 on theopposite sides with the tips 3 therebetween, each have one permanentmagnet block 35 out of eight provided for the supporting section 34.

The linear magnetic sources 36 and 37 are not only provided so as to beable to come closer or go away (refer to FIG. 7 and FIG. 8) with respectto the tip 3 as shown by the horizontal arrows in FIG. 9, but also so asto be movable vertically as shown by the arrows along the axialdirection of the respective tips 3. Moreover, the tip 3 itself isrotatable about the axis as shown by the curved arrow.

Furthermore, it is possible to apply the magnetic force in a route asshown by the curved broken lines of the drawing, by moving therespective linear magnetic sources 36 and 37 along both of the axialdirection and the close/away direction of the tip 3 at the same time. Inthese examples, various magnetic threes are applied to the inside of thetip 3 exclusively by moving the linear magnetic sauces 36 and 37 of themagnetic three treatment unit 27. However, it is also possible to applythe same type of magnetic force by moving the nozzle unit 17 itself. Inthis case, the linear magnetic sources 36 and 37 correspond to themagnetic source, and the transfer unit of the nozzle unit corresponds tothe magnetic source transfer unit.

According to the present embodiment, since the magnetic force is appliedto the tips being the respective packing units, from at least twodirections, it is possible by the magnetic force to avoid the conditionwhere the magnetic supports are attached to or precipitated on the wallor the bottom of the packing unit respectively. By moving the linearmagnetic sources vertically and horizontally, or by rotating therespective tips, the magnetic supports can be moved relatively withrespect to the host in a state where they are developed in a higherdensity than the other areas within the packing unit, thus increasingthe number of collisions or the rate of collisions of the magneticsupports and the host, or increasing the chance of encounter, so thatthe introduction treatment can be performed efficiently.

In the above examples, the operation of the packing unit, the pressureadjuster, the magnetic source, the magnetic force transfer unit, thetransfer unit and the like is controlled by an information processor(not shown). The information processor has: a CPU; a memory, a CDROM, aflexible disk, a DVD and the like which store various programs such asthe procedure for the introduction treatment or various data on thebiological material, the host, the magnetic support, the magneticsource, the environmental conditions, and the like; input devices suchas a keyboard and a mouse for inputting various data such as host data,biological material data, reagent data, and the like, and instructionsof various operations; output devices such as a display device and aprinter which show the result of introduction, treatment status, therate of collision, the number of collisions, or the probability ofencounter (density or distribution of magnetic support or host); and acommunication facility such as a modem.

Next, FIG. 10 shows seven types of magnetic supports according to athird embodiment.

The first type of magnetic support shown in FIG. 10 (1) is a globularparticle, having a size between 100 nm and several μm for example, whichis used if the particle surface coat and the host have the good adhesiveproperty. For the material, a magnetizable superparamagnet is used,which is made of for example, an iron hydroxide, an iron oxide hydrate,an iron oxide, a mixed iron oxide, or iron.

The second type of magnetic support shown in FIG. 10 (2) has a majoraxis where one axis is formed longer than the other axis, being arotating symmetry body with respect to the major axis, and the oppositeends along the major axis are formed tapered. If it is magnetized, orformed to be magnetizable, in the major axis direction, it becomesmovable by the magnetic force in the major axis direction, and thus isreadily controlled. The size of the magnetic support is about between100 nm and several μm for example, and the material is similar to thatdescribed for the first type of magnetic support. In the presentexample, the magnetic support has the structure for facilitating entryinto the host, and is used for example in a case where there is a cellwall, or the cell membrane is silting, or a case where cell surfacetreatment is not performed.

The third type of magnetic support shown in FIG. 10 (3) has a poroussurface, or carries other porous material. Accordingly it can reliablycarry the biological material.

The fourth type of magnetic support shown in FIG. 10 (4) is globularhaving a large number of projections on the surface of the magneticsupport itself or the material carried by the magnetic support.Accordingly, it can reliably carry the biological material betweenprojections.

The fifth type of magnetic support shown in FIG. 10 (5) has a pluralityof, for example the first to fourth types of the magnetic supportsconnected. The connection portion is used as the carrier. The magneticsupport is readily produced and is of low price.

The sixth type of magnetic support shown in FIG. 10 (6) has a major axislonger than the other axis, being a rotating symmetry body with respectto the major axis, and the opposite ends along the major axis are formedtapered. Moreover, a carrier having an annular groove on the side faceis provided. As a result, the capacity to carry the target biologicalmaterial on the magnetic support can be increased. The material issimilar to that of the first type of magnetic support for example.

The seventh type of magnetic support shown in FIG. 10 (7) is a complexsupport being, for example a globular particle-shaped object combinedwith filamentary supports as a carrier. It is used, for example in acase where the cell membrane is relatively weak, requiring introductionunder a mild condition, in a case of a dilute fungus liquid, or in acase where handling is facilitated if the target biological materialsare aggregated. The respective filamentary supports are not introducedinto a host, but a load is applied to the hosts entwined with thefilamentary supports by applying the magnetic force, so that the forceis applied as a line, and not as a point, to reduce damage. Since thismethod allows treating the particle and the carrier separately,manufacture is facilitated.

If the target biological material is adhered to or combined with themagnetic support by physical adsorption or electrical interaction, theyare readily separated inside the host. Moreover, the magnetic supportmay carry an introduction adjuvant for facilitating introduction of themagnetic support into the host. For example, when a bacterial gene isintroduced, the membrane fluidity is increased by calcium chloride so asto facilitate taking in of the plasmid. In a case of an animal cell, itis protoplastid using polyethylene glycol and then introduced. Moreover,in order to increase the chance of contact with cells, cell aggregationmay be promoted (crosslinking between cells by bivalent metal ions andthe like) so as to perform the introduction treatment in a conditionwhere the cells are conglomerated.

Next is a description of a gene therapy using the apparatus forintroducing a biological material and the method of introducing abiological material according to a fourth embodiment, based on FIG. 11.

As shown in FIG. 11, in step S1 cancer cells collected turn a cancerpatient are placed in a container. A contrast medium such as Fe (Cu)having a property of attaching to the cancer cell tissues, isadministrated to the vicinity of the cancerous area by injection, thecancer cells are collected using X ray irradiation, and then put intothe solution packing unit 24 of the container 25. In step S2, themagnetic supports and proteins p53 are mixed in the solution, andrepeatedly drawn and discharged lightly by the nozzle unit 17, so thatthe magnetic support can carry the protein p53.

Here, “p53” is one type of protein which monitors the presence/absenceof DNA damage in a cell at all times, and allows DNA replication only ifthere is no DNA damage. Moreover, when it finds an abnormality in theDNA, it inhibits transcription by a transcription factor and stops thereplication of the damaged DNA, and the large number of the produced p53repair the DNA damage rapidly. If the damage is peat at this time, itgives up repairing the cell, and gives instructions to remove the cell.

Therefore, by introducing p53 into a cancer cell, there is provided aproperty of stopping the replication of the cancer cell, and repairingthe DNA damage rapidly.

In step S3, a PEG (polyethylene glycol) solution corresponding to theadjuvant, and the magnetic supports carrying p53 being the biologicalmaterial, are mixed in a predetermined solution packing unit of themicroplate 21, and lightly stirred by repeatedly drawing and dischargingusing the nozzle unit 17 so that the magnetic support can carry the PEG.Here, the PEG solution is to soften the cell membrane so that themagnetic support can enter readily.

In step S4, the cancer cells, and the magnetic supports carrying the p53and the PEG are mixed in the solution to make the mixture solution inthe solution packing unit of the microplate 21.

In step S5, the nozzle unit 17 is moved to the solution packing unit ofthe microplate 21 containing the mixture solution, and the mixturesolution is drawn as far as the storage unit 6 of the tip 3.

In step S6, the nozzle unit 17 by which the mixture solution is drawn,is moved to the magnetic three treatment unit 27 while carrying themixture solution, and the tips 3 are inserted into the openings of themagnetic three treatment unit 27.

Next, the linear magnetic sources 36 and 37 in the magnetic forcetreatment unit 27 are brought close to the tips 3 arranged in a linearform, and are moved by about the width of the tip 3 in parallel with theline, or are moved vertically. By so doing the magnetic supports aredeveloped in the tip 3 and are moved within the tip 3 in the developedstate, so as to increase the number of collisions or the rate ofcollisions of the magnetic supports and the cancer cells being the host,or increase the chance of encounter thereof thus making the magneticsupport enter the cancer cell.

In step S7, after the p53 is introduced into the cancer cell by makingthe magnetic support enter the cancer cell, one linear magnetic source36 in the magnetic three treatment unit 27 is brought close to thenozzle unit 17 and the other linear magnetic source 37 is taken awayfrom the nozzle unit 17 for a predetermined distance or more. Thereby aunidirectional magnetic force is applied to the magnetic support, so asto separate the cancer cells which the magnetic supports enter, bymaking them attach to the inner wall of the tip 3. After discharging theresidual liquid, the separated cancer cells are moved to the container29 together with the nozzle unit 17, while being attached to the innerwall of the tip 3. By repeatedly drawing and discharging the solution inthe container 29, the cells are resuspended in the solution, which isthen put into the solution packing unit.

In step S8, the solution containing the separated and extracted cellswhere the abnormality of the DNA has been repaired by the action of theintroduced p53, and into which the magnetic support has been entered, isagain drawn and held by the nozzle unit 17. Then, the solution is movedto the reagent bath 18 and discharged to the media therein and the cellsare cultured.

In step S9, the solution containing the repaired cells into which thep53 is introduced, is again drawn and held by the nozzle unit 17 andmoved to the magnetic three treatment unit 27. One linear magneticsource 36 is brought close to the nozzle unit 17 and the other linearmagnetic source 37 is taken away from it. Therefore the repaired cellswhich the magnetic supports have entered into and remain inside, areseparated and removed by making them attach to the inner wall of the tip3. The residual solution is discharged and put into the solution packingunit 28 of the container 29. The used tip 3 having the absorbed cellscontaining the magnetic supports is detached and disposed into the wastevent 30.

In step S10, the repaired cells into which the p53 obtained in this wayby pure culture has been introduced, are adhered to Fe (Cu) having theproperty of attaching to the cancer cells, and then administrated to thediseased area of the patient by injection. When cells having p53introduced are administrated in large amounts, the cancer cells can beremoved or repaired due to the action of the p53, enabling treatment ofthe cancer.

Next is a description of a production method for a protein according toa fifth embodiment, based on FIG. 12. This method shows an example wherea magnetic force is not required for separation.

In step S11, a recombinant plasmid having a target gene to produce auseful protein to be synthesized inside a cell is made. Regarding therecombinant plasmid (Pbr322), a ring-shaped plasmid is cut by adding arestriction enzyme and is made into a linear-shape, then mixed with thetarget gene DNA, and a DNA ligase is added thereto to make thering-shaped recombinant plasmid.

In step S12, a large number of the recombinant plasmids are mixed with alarge number of the magnetic supports in a solution, so as to make themagnetic support carry the recombinant plasmid.

On the other hand, in step S13, a solution containing E. colis as a hostis drawn from a container in which it is contained using the nozzle unit17. In step S14, it is moved into a container containing a surfacetreatment reagent as the introduction adjuvant which softens the surfaceof the E. coli, then discharged and mixed.

In step S15, a mixture solution is made by mixing E. colis and themagnetic supports carrying the recombinant plasmid in the solution whilegently stirring, and put in the solution packing unit 20 in themicroplate 21 in FIG. 4.

In step S16, the microplate 21 is cooled during incubation (coolingtemperature is 4° C.) in the heated/cooled area 31, so as to recover thecells which were damaged during mixing with the reagent or duringmovement.

In step S17, in order ID facilitate the introduction of the plasmid asthe target biological material carried by the magnetic support into theE. coli, heat shock is applied by heating for 30 to 90 seconds at 42° C.in the microplate 21 in the heated/cooled area 31.

In step S18, the nozzle unit 17 is moved to the microplate 21, and thento the magnetic force treatment unit 27 while drawing and holding themixture solution, and the linear magnetic sources 36 and 37 are moved ina predetermined direction, so as to apply the magnetic three to thenozzle unit 17. As a result, in the state of being developed the mixturesolution, the magnetic supports are moved relatively with respect to thehost by the magnetic force, so as to increase the number of collisionsor the rate of collisions, or increase the chance of encounter, makingthe magnetic supports enter into the E. coli.

In step S19, the introduction treated mixture solution is moved to themicroplate 21 while being drawn into and held in the respective tips 3of the nozzle unit 17, and discharged thereto. Then, the mixturesolution is cooled down quickly to 4° C. by incubation in theheated/cooled area 31, and the cell membrane of the E. coli is closed tocomplete the introduction treatment.

In step S20, in order to separate and extract the E. colis into whichthe plasmid with the target biological material is introduced, anantibiotic is added to the mixture solution for which the introductiontreatment is completed, so as to kill the E. colis not having therecombinant plasmid introduced, and the E. colis into which the plasmidis introduced are separated.

In step S21, the mixture solution containing the E. coli into which theplasmid is introduced, is drawn and held by the nozzle unit 17, thentransferred to the medium of the reagent bath 18, and discharged to themedium.

The mixture solution discharged to the medium is cultured by incubationat 37° C. for 30 to 60 minutes and the host cells are repaired andstabilized.

The above respective embodiments have been specifically described forbetter understanding, and are not to be considered as limiting otherembodiments. Therefore, modifications can be made without departing fromthe scope of the present invention. For example, in the descriptionabove, only a case where an annular magnet or two linear magnets withthe tip therebetween, are used, has been described. However, theinvention is not limited to this example, and for example permanentmagnet blocks or electromagnets may be used in some cases.

Furthermore, in a configuration where three or more of the permanentmagnet blocks or the electromagnets are arranged so as to have the samecentral angle as each other, around the packing unit of a circulartubular shape, a magnetic force modification unit which modifies therespective magnetic forces sequentially at a predetermined period, maybe provided instead of the magnetic source transfer unit.

Alternatively, the magnetic source transfer unit may have as themagnetic source, three or more of the permanent magnet blocks orelectromagnet blocks which are arranged so as to have the same centralangle as each other, around the packing unit of a circular tubularshape, so that the magnetic source can be made radially closer to orapart from the packing unit or can be movable along the axial directionand the circumferential direction. Moreover, in the magnetic forcetreatment unit, a plurality of annular permanent magnets may be arrangedinstead of the linear magnetic sources. Furthermore, the linear magneticsources may be provided for the packing unit.

Moreover, the two magnets or the magnetic poles provided so as to faceeach other, of the linear magnetic sources may have the same pole oropposite poles. Furthermore, it is preferable in terms of the structuralstability of the apparatus, that the permanent magnet blocks arranged onthe linear magnetic source are arranged to have different magnetic polesalternately, and the facing linear magnets are provided with magneticpoles different to the corresponding magnetic poles.

Only the case where the linear nozzle unit has a set of eight, has beendescribed, however the number is not limited to “eight” and may beselected based on the number of container wells of the microplate to beused. The packing unit is not limited to the illustrated tip but hasvarious shapes. The host and biological material are also not limited tothe examples of the embodiments and have various types. Moreover, theplurality of packing units may be arranged not only in a linear form butalso in a matrix form. In this case, the magnetic sources for theintroduction treatment units are arranged in a matrix form correspondingto the arrangement of the packing units. Therefore, the integration canbe further advanced and the introduction treatment can be furtherefficiently performed. The arrangement may be not only a linear form nora matrix form, but in a curvilinear form.

Next is a description of an apparatus for introducing a biologicalmaterial according to a sixth embodiment, based on FIG. 13 and FIG. 14.The same reference symbols are used for components the same to thosedescribed in the second embodiment, and the description thereof isomitted.

The apparatus for introducing a biological material according to thesixth embodiment is provided with a perforation treatment unit whichperforates the cell membrane or the like, being the boundary, from theoutside of the host such as the cell within the packing unit. Theperforation treatment unit has a perforation force source provided atthe tip corresponding to the packing unit in order to apply aperforation force to the host, and a perforation force source controlunit which controls the perforation farce source. Examples of theperforation force source are described based on the drawings.

FIG. 13 (1) shows a nozzle unit 39 wherein the set of eight nozzles 2described in the second embodiment are arranged with their respectiveaxes vertical and in parallel with each other at intervals along thehorizontal line, and tips 40 are detachably inserted into the nozzles 2respectively. Moreover, as described in the second embodiment, the setof eight nozzles 2, and therefore the tips 40, are arranged along thehorizontal line. The magnetic force treatment unit has two linearmagnetic sources 36 and 37 which have rod-shaped supporting sections 34provided on the opposite sides with the tip 3 therebetween, in parallelalong the horizontal line, and eight permanent magnet blocks 35 set inpositions corresponding to the respective tips 3.

In FIG. 13 (1), as the per titration force source to generate dischargepulses, a pair of facing electrodes 41 and 42 are provided on the innerwall of the small diameter liquid passage 5 of the tip 40 at a heightcorresponding to the linear magnetic sources 36 and 37. Conducting wires43 and 44 reach from the respective electrodes 41 and 42 through themiddle diameter unit 7 and the inner wall of the large diameter storageunit 6 to the attachment unit 8 along the axial direction. The ends ofthe respective conducting wires 43 and 44 are to be connected toterminals 45 and 46 which are connected with a power circuit (not shown)provided on the outer surface of the nozzle 2 via conducting wires 47and 48, at the time of attachment. By applying a predetermined dischargepulse from the power circuit to between the pair of the electrodes 41and 42 in a state where a magnetic force is applied by the linearmagnetic sources 36 and 37, the perforation treatment is applied to thehost passing through the liquid passage 5.

In FIG. 13 (2), a nozzle unit 49 is used instead of the nozzle unit 39described in FIG. 13 (1).

In the nozzle unit 49, as the perforation force source to generate thedischarge pulses, a pair of facing electrodes 51 and 52 are provided onthe inner wall of the large diameter storage unit 6 of the tip 50 at aheight corresponding to the linear magnetic sources 36 and 37.Conducting wires 53 and 54 are extended from the respective electrodes51 and 52 along the inner wall of the storage unit 6 upward reaching theattachment unit 8. The ends of the respective conducting wires 53 and 54are to be connected to terminals 55 and 56 which are connected with thepower circuit (not shown) provided on the outer surface of the nozzle 2via conducting wires 57 and 58, at the time of attachment. By applying apredetermined discharge pulse from the power circuit to between the pairof the electrodes 51 and 52, the perforation treatment is applied to thehost pooled in the storage unit 6.

According to the tip 39 in FIG. 13 (1) or the tip 49 in FIG. 13 (2), itis possible to apply the voltage to the electrodes, being theperforation treatment unit, simply by attaching the tip 39 or 49 to thenozzle 2, thus facilitating the handling.

In FIG. 14 (1), a nozzle unit 59 is used instead of the nozzle units 39and 49.

In the nozzle unit 59, as the perforation force source to generate thedischarge pulses, a pair of facing electrodes 61 and 62 are provided soas to pass through the wall of the large diameter storage unit 6 of thetip 60 from the inside to the outside at a height corresponding to thelinear magnetic sources 36 and 37. In order to apply the voltage to theelectrodes, they may be connected to the power circuit via portions 63and 64 exposed to the outside of the electrodes. Therefore, since it isnot necessary to pass the conducting wire to inside the tip 60, which isnecessary in the nozzle units 39 and 49, the internal structure can besimplified and the manufacturing cost can be reduced. Moreover, sincethe inside of the tip 60 is not so uneven, the flow of a fluid in thetip 60 can be smoothened.

In FIG. 14 (2), a nozzle unit 65 is used instead of the nozzle units 39,49, and 59.

In the nozzle unit 65, as the perforation force source to generateultrasound, an ultrasonic oscillator 67 is provided at the heightcorresponding to the linear magnetic sources 36 and 37. A conductingwire 58 is provided from the ultrasonic oscillator 67 along the axialdirection inside the tip 66, and it is provided so that the end of theconcluding wire 58 is connected to a terminal provided on the outersurface of the nozzle 2, at the time of attachment of the tip 66.

The perforation force source control unit which controls the perforationforce source comprises an information processor and a program providedin the information processor. The perforation force source control unitcontrols the perforation force based on the properties, the amount, orthe density of the host, the biological material to be inserted into thehost, or the magnetic support, and on the magnetic force control unitwhich controls the linear magnetic sources 36 and 37. That is, regardingthe perforation into the host, the intensity of the perforation force isdetermined by the hardness of the host, the size of the magnetic supportto be entered, or the acceptable size to introduce the biologicalmaterial fixed on the magnetic support when the magnetic support isattached to the host. Moreover, the generation lime, the frequency andthe like of the perforation force are determined based on the amount andthe density of the host or the magnetic support.

The perforation force source control unit generates the perforationforce in spatial or time association with the introduction treatment,according to the properties of the host. For example, if the host is acell, when the cell membrane is perforated, it is repaired in a shorttime. Therefore it is necessary to execute the introduction treatment atthe same time as the perforation treatment, or to execute theperforation treatment immediately after the introduction treatment. Forthat reason, the introduction treatment perforation treatment arerequired to be executed in spatial or time association with each other.

According to the present embodiment, the biological material carried bythe magnetic support can be efficiently introduced into the host bycombining and controlling the electric field and the magnetic field.

Next is a description of a seventh embodiment.

In FIG. 15 (1), there we a microplate 70 having eight holes 71 intowhich tips can be inserted all at once using the set of eight nozzlesunit 17 (or 39, 49, 59, and 65), and a separation unit 73 on which themicroplate 70 is mounted and which has a plurality of permanent magnetblocks 72 arranged at positions corresponding to the bottoms of therespective holes 71. Media are contained in the respective holes of themicroplate 70.

The introduction treated (or introduction treated and perforationtreated) solution containing the host having the introduced magneticsupport, is drawn using the set of eight nozzles unit 17, thentransferred to the microplate 70 having the holes 71 containing themedia, while being contained in the respective tips 3 (39, 49, 59, and65), by moving the nozzle unit 17 itself, and then discharged into therespective holes 71 containing the media, all together.

The host is cultured by incubation in the respective holes 71 for apredetermined time at a predetermined temperature, and the firstgeneration host contained first and having the introduced magneticsupport is moved to the bottom of the respective holes 71 by themagnetic force of the permanent magnet block 72.

Then, as shown in FIG. 15 (2), by drawing only a portion which is notattached to the bottom in the respective holes 71 using the newlyattached tip 74, while the linear magnetic source 36 (37) is close tothe liquid passage, the pure host having the introduced and culturedbiological material can be extracted.

According to the present embodiment, a pure cultured substance can beextracted efficiently and reliably with a simple apparatus.

Moreover, the abovementioned respective components, parts, units, forexample, the packing unit, the container, the magnetic source, theliquid passage, the filter holder, the nozzle, the tip, the magneticsource transfer unit, the magnetic force modification unit, thepermanent magnet, the electromagnet, the magnetic support, the carrier,the reagent, the host, the biological material, the magnetic forcetreatment unit, the introduction treatment unit, the transfer mechanism,the perforation treatment unit, the perforation force source (electrode,ultrasonic oscillator), the conducting wire, the terminal and the likemay be optionally combined while appropriately modifying them ifrequired. Particularly, the third to the eighteenth aspects of thepresent invention may be applied to the packing unit and theintroduction treatment unit of the thirty-first aspect of the presentinvention, and the twentieth to the twenty-seventh aspects of thepresent invention may be applied to the mixing step and the introductiontreatment step of the thirty-seventh aspect of the present invention.

Moreover, the arrangement may be such that, for example the unusedpacking unit, the liquid passage in a tip form, the liquid passage in atip form with a filter, the filter holder, and the like are stored in arack, making them attachable by moving the carrier of the packing unit,the nozzle of the pressure adjuster, or the liquid passage by thetransfer mechanism, or that these used carrier, the liquid passage, andthe like are detached and a rack to store them is provided. Therefore,the introduction treatment can be standardized and automaticallyperformed without relying on manpower. The transfer mechanism includingthe packing unit containers provided outside, liquid passage, themagnetic source, and the nick, may be movably provided.

The mechanism to realize the apparatus for introducing a biologicalmaterial according to the first embodiment has not been specificallydescribed. However, it can be realized by applying the mechanismdescribed in the biological material introduction system according tothe second embodiment, to one nozzle only.

Moreover, only the case where the perforation treatment unit is appliedto the set of eight nozzles unit has been described, however it is alsoapplicable to one nozzle unit only, and the number of the nozzles in thenozzle unit is not limited to eight. The respective mechanisms, therespective parts, the forms, and the like shown in the respectiveembodiments described above, show only examples and are not limited bythese examples. Moreover, the introduction treatment may be performedafter increasing the permeability of the boundary of host in order toincrease the efficiency to introduce the biological material. For thatpurpose, for example, the introduction may be performed after inducingthe host by a divalent ion such as Ca, or after inducing the host by anorganic solvent in some cases.

1. An apparatus for introducing a biological material comprising; one ormore packing units containing a mixture solution having a plurality ofmagnetic supports carrying a biological material to be introduced into ahost such as a cell and having a size allowing entry into said host, anda plurality of said hosts in a solution; a perforation treatment unitwhich perforates said host; and an introduction treatment unit whichmoves said magnetic supports relatively with respect to said hosts inthe solution by controlling a magnetic force applied to inside saidpacking unit from at least two directions with said packing unittherebetween so as to introduce said biological material into said hostsby encounter between the magnetic supports and the perforated hosts. 2.An apparatus for introducing a biological material according to claim 1,wherein said introduction treatment unit comprises a magnetic sourcewhich can apply a magnetic force to the inside of said packing unit; anda magnetic force control unit which controls relative movement betweensaid magnetic supports and said host by changing the relative positionor the velocity between said packing unit or said mixture solution andsaid magnetic field, or the magnetic force itself due to said magneticsource.
 3. An apparatus for introducing a biological material accordingto claim 1, wherein said perforation treatment unit has: a perforationforce source which can apply a perforation force by an electric field,or ultrasound, or the like; and a perforation force source control unitwhich controls said perforation force source.
 4. An apparatus forintroducing a biological material according to either one of claim 1 andclaim 2, wherein said perforation force source control unit controls theperforation force source based on the properties, the amount, or thedensity of said host, said biological material, or said magneticsupport.
 5. An apparatus for introducing a biological material accordingto any one of claim 1, wherein said perforation force control unit orsaid magnetic force control unit control the introduction treatment andthe perforation treatment so as to be executed in spatial or timeassociation with each other.
 6. An apparatus for introducing abiological material according to any one of claim 1, where said packingunit has a liquid passage through which said mixture solution can pass,and has a pressure adjuster which draws and discharges the solution byadjusting the pressure in said liquid passage, as said magnetic forcecontrol unit.
 7. An apparatus for introducing a biological materialaccording to claim 1, wherein said introduction treatment unit movessaid magnetic airports and said host relatively to each other in a statewhere a plurality of said magnetic supports in the solution contained insaid packing unit are developed in solution by the magnetic force, andcontrols so as to move said magnetic supports.